The production of force in muscle cells as well as in many non-muscle cells is the result of the interaction of ATP with the two proteins actin and myosin. My proposed research will be concerned generally with the elucidation of the molecular mechanism of the actomyosin interaction and in particular with the role of actin. The studies will concentrate initially on an actin bound nucleotide which is dephosphorylated during the polymerization of the actin, but whose function remains unknown. I will study this problem with the use of two analogs of ATP which can replace the native nucleotide. An unsplittable analog will be used to prevent the dephosphorylation. The interactions of the actin will then be monitored to determine which are inhibited by the elimination of the dephosphorylation. I have already determined that the polymerization itself is not inhibited, but the presence of the analog does inhibit some actomyosin interactions. I will also incorporate a fluorescent analog of ATP into the actin site to look for some interaction of actin with other proteins which may affect the environment of the bound nucleotide. Additional studies will investigate fluorescent energy transfer between the analog bound to the actin and other fluorescent probes bound to tropomyosin or myosin. These studies will be aimed at determining distances between sites on the contractile proteins, and thereby monitoring protein movements and conformation changes. A related project will study the properties of actin isolated from amoeba. The objective of this study is to understand the regulation of the actin polymerization in vivo in the cytoplasm of the amoeba.